Aerolysin, a Powerful Protein Sensor for Fundamental Studies and Development of Upcoming Applications

Nanopore electrical approach is a breakthrough in single molecular level detection of particles as small as ions, and complex as biomolecules. This technique can be used for molecule analysis, and characterization as well as for the understanding of confined medium dynamics in chemical or biological reactions. Altogether, the information obtained from these kinds of experiments will allow to address challenges in a variety of biological fields. The sensing, design and manufacture of nanopores is crucial to obtain these objectives. For some time now, aerolysin, a pore forming toxin, and its mutants have shown high potential in real time analytical chemistry, size discrimination of neutral polymers, oligosaccharides, oligonucleotides and peptides at monomeric resolution, sequence identification, chemical modification on DNA, potential biomarkers detection and protein folding analysis. This review focuses, on the results obtained with aerolysin nanopores on the fields of chemistry, biology, physics and biotechnology. We discuss and compare as well the results obtained with other protein channel sensors. two main that control neutral to charged macromolecules: electro-osmosis

For the past two decades, the number of papers and the citations in the field of nanopores has increased exponentially. Nanopore technology has become a sensitive, selective, low cost, label-free, real-time and transportable tool for sensing a wide variety of molecules, including ions, polymers, polyelectrolytes, viruses, ligand-molecule complexes and biomolecules. It allows for the analysis of transport properties, conformations, folding, size, sequence or chemical modifications 1-6 . At first, the protein channels were the main sensors used to perform numerous studies [7][8][9] . Thanks to material science, chemistry, nanoscience and molecular biology, it is now possible to design and manufacture new classes of nanopores: solid-state sensors 10,11 , DNA origami nanopores 12,13 , carbon 14,15 or cyclodextrin nanotubes [16][17][18] , hybrid nanopores [19][20][21] and glass 22,23 or quartz nanopores 24 with DNA aptamers 25 . The increased interest in nanopore research has been mainly associated to the ultra-fast DNA sequencing challenge, recently achieved by Oxford Nanopore Technology [26][27][28][29] . The objectives at the horizon of this field comprise of proteomic sequencing 6,[30][31][32][33] , biomarker detection (of micro-RNAs 34 as well as infinitesimal peptides and proteins quantities) and single-molecule mass spectrometry 32,[35][36][37][38][39][40][41][42] . Up to now, the best sensitivity for biotechnological or heath applications is obtained through biological channels.
A member of the pore-forming toxin (PFTs) 43 family, aerolysin is a beta structure toxin that has recently been at the center of extensive fundamental studies and some biotechnology applications 32,38,39,[44][45][46][47][48] . In this review we first introduce the molecular mechanism of channel formation, from soluble inactive monomers to a functional pore into a lipid membrane (figure 1); we delve into the structure of monomeric and heptameric aerolysin along with, the mechanism promoting transition between the pre-pore and the fully functional pore according to the latest findings in X-ray crystallography, cryo-EM, molecular dynamics and computational modeling (figure 1). The ability to obtain single molecule resolution information from electrical signals, depends on efficient data analysis and amplifier quality. We present the principle of electrical molecule detection, the set-up, the acquisition and filter effects, the background for data analysis from current traces to obtain statistical distributions of events along with their duration, frequency and current amplitude (figure 2). One of the challenges of nanopore single molecule sensing is dynamics control, entry and interactions between the molecules and the pore. We focus on the two main driving forces that control neutral to charged macromolecules: electro-osmosis and electrophoresis. These forces depend on aerolysin selectivity, applied voltage, temperature, pH, salt concentration and salt make-up (figure 3, 4). We also look at aerolysin's powerful sensor competence noted by its ability to detect different protein conformations, stability and unfolding transitions ( figure 5). Furthermore, we show how aerolysin allows for size discrimination with a monomeric resolution or sequence determination of oligosaccharides, polymers (figure 6), oligonucleotides (figure 7) and peptides (figure 8). We discuss the best discrimination resolution obtained with aerolysin. We conclude this review with a brief discussion of the major results obtained with aerolysin in comparison with other protein sensors and the potentials for upcoming applications.

I. Aerolysin channel and electrical detection
Biological nanopores are a large family of proteins and peptides that are implicated in many biological processes. Some of these nanopores have been intensively used in studies where the nanopore molecule inserted in a lipid bilayer is coupled to an electrical detection system allowing single molecule characterization 3, 8 . Aerolysin is one of the most promising nanopores for biotechnology applications 45 . This protein is a β-barrel pore forming toxin (β-PFT) that is implicated in pathogenic infections by Aeromonas 47 . Bacteria produce this protein as a precursor protein, pro-aerolysin, which forms soluble dimers 48 . At membrane proximity, a proteolytic cleavage of the pro-peptide sequence allows oligomerization of the aerolysin to form a heptameric pore 49,50 . Monomers first assemble into a heptameric pre-pore structure. This complex docks onto the membrane surface, with the pre-stem loops ready to slide through a pocket into the interior cavity of the pre-pore. Triggering the transition from pre-pore to pore, the pre-stem loops eventually refold into amphipathic β-hairpins forming the transmembrane β-barrel. This conformational change is accompanied by a concerted swirling mechanism that flattens the extracellular portion of the pore as the β-barrel forms and inserts into the membrane 43 ( Figure   1A). Aerolysin heptamers show a mushroom-like structure with a β-barrel stem inserted in the lipid membrane ( Figure 1B middle). Extracellular structure has not a real vestibule as ⍺hemolysin but there is a cap with a collar structure that is 16 nm large. The internal pore diameter is 1.8 nm ( Figure 1B, left and middle). One of the most important characteristics of an aerolysin pore for electrical coupled detection of single macromolecules is charge distribution ( Figure 1B,   right). Surprisingly, the β-barrel is composed of an alternation of positive and negative amino acids (lysine and glutamic acid) Site-directed mutagenesis experiments have been developed to change selectivity and sensitivity of aerolysin nanopore and contribute to potential biotechnological application of this pore protein 45 .
Obtaining the tri-dimensional structure of an aerolysin nanopore has been far from an inconsequential task. The crux of the problem lies in the original insertion mechanism of this protein pore. Indeed, protein domain organization changes drastically between pro-aerolysin soluble dimers and the heptameric nanopore conformation ( Figure 1C). Intermediates between both states have also been identified by combining classical X-ray crystallography, cryo-EM, molecular dynamics and computational modelling 46 .
The applications of aerolysin as a channel forming protein that can oligomerize to form a heptameric structure into lipid bilayers are developed since 1990s [51][52][53] . This toxin is synthetized as an inactive precursor, pro-aerolysin 52 kDa, that stabilize as dimers 54 (PDB code: 1PRE, Figure 1C left). Activation is initiated by C-terminal cleavage of the pro-peptide sequence (25 amino acids) followed by oligomerization of the toxin protein 50 . The first low resolution (25 Å) structural studies of membrane inserted aerolysin where obtained by Parker and colleagues 48 .
After these studies, development of a mutant version of aerolysin 55 allowed to further understand the heptameric structure of the channel. This mutant assembled into a hydrophilic oligomer with a mushroom-shaped structure akin to its wild type counterpart. The combination of X-ray crystallography, cryo-EM, molecular dynamics and computational modelling was necessary to achieve the structure of the membrane inserted nanopore 46 (PDB: 5JZT). In this study, several transition structures were identified due to a reorganization of the pre-stem loop between domains 3 and 4 after proteolytic cleavage of the pro-peptide sequence at the lipid membrane proximity that finally contributes to create a transmembrane β-barrel ( Figure 1C  Understanding pore insertion mechanism has been essential for other pore forming toxins of the aerolysin family such as lysenin or epsilon-toxin 56 . Other aerolysin-like, membrane pore-forming proteins whose soluble structure where well understood have been only recently described and show similar assembly mechanisms to the above described 57 . Furthermore, aerolysin has been shown to be resistant to high concentration of denaturing agent (up to 1.6 M guanidium) 58 , ample pH (from pH 2.1 to pH 10) 39,59 and high temperatures (up to 70°C) 60,61 , making this a durable pore with high potential for biotechnological applications. First, we consider one electrolyte (usually 1M KCl) filled compartment, which is divided in two sub-compartments (cis and trans) by a lipid bilayer 2,62-65 . This membrane behaves as an insulating wall, in which a single aerolysin channel is inserted. This insertion is followed by measurement of the ionic current through the protein channel in the presence of an electrical potential ΔU between the ground (cis compartment) and the reference electrode  Figure 2b). The channel conductance G p is defined as the ratio between the ionic current I 0 and the applied voltage ΔU: G p = I 0 /ΔU = 0.56 ± 0.05 nS. In a first approximation, we assume the aerolysin behaves as a conducting cylinder of radius R and length ℓ:

Principle of molecule detection by electrical measurement and data analysis
where Κ b is the bulk conductivity of the electrolyte 11 . In 1M KCl, G p = 1.9 nS. This value is overestimated because we neglect the ion confinement in the aerolysin channel 17,66 . This relation is true in high ion concentration, when the conductance is mainly due to the ionic flow through the channel. On the other hand, if the concentration is too low, the thickness of the counter ion layer at the inner surface of the channel is larger than the channel radius 11,67,68 . This thickness is controlled by the screening Debye length λ D . 69 Here, the lipid bilayer behaves as an insulating membrane and is characterized by its capacitance C m , which is in parallel with the pore conductance G p and can be approximated by the following equation: where ε rm = 2 is the dielectric constant of the membrane capacitance, A m the area and ℓ m the thickness of the membrane. Typically, by taking the membrane size Ø 90 µm into account, C m is larger than 65 ± 5 pF leading to an estimated membrane thickness 70 of about 4.85 ± 0.4 nm. This thickness is low enough to allow easier channel insertion. Now, we consider the access conductance G cis and G trans , which correspond to the conductance between each electrode and the corresponding entrance of the channel. They are evaluated by the formula 71 : Then, we can define the overall access conductance G acc = G cis G trans / (G cis + G trans ) : In 1M KCl, this formula leads to a G acc value of 36 nS. This expression is applicable to neutral where the G m = s C m is the complex conductance due to the membrane capacitance in the Laplace space 73 .
After addition of deca-saccharide chains of hyaluronic acid, we observe current blockades 70,74 ( Figure 2b). Given that there is no correlation between each blockade, the statistical distribution of the duration between two following blockades T i follows a Poisson's law, defined by an exponential decay ≈ exp (f T i ) (Figure 2c), where f is the characteristic frequency. This frequency is controlled by the confinement energy of the chain into the channel 2,58,75-77 . If the chain is charged, this energy could be lowered by the addition of electrostatic energy 58,78 . When the chain enters the nanopore, we measure a current blockade characterized by its duration or dwell time T t and its amplitude ΔI b (plotted in the scatter plot in figure 2e). These experimental values could be strongly modified both by the current amplifier, which measures the ionic current, and the acquisition card used for data processing.
The low-pass filter integrated in the amplifier could smooth a blockade, if its duration is shorter than the double of the rise time T r of the filter defined by: 79 T r = 0.33/f c (6) Assuming f c = 10 kHz, blockades must be shorter than 2T r ≈ 66 µs to avoid deformation but some algorithms have been developed to overcome this limitation 73,79 . Considering Shannon's theorem, the sampling time T s of the acquisition must be two times smaller than the characteristic The blockade current histogram is characterized by one peak, which is fitted by a Gaussian function (figure 2g). Because the blockades' amplitude being a function of the chain length, this distribution shows that the deca-saccharide chain is monodisperse 32,38,67,82,83 .

II.
From the conformation to the monomer sensing

c) Effect of temperature
The effect of temperature on oligonucleotides transport through aerolysin has been explored by Payet et al, especially its influence on the entrance and transport energy barrier 60 . The authors describe an Arrhenius type temperature dependence for the frequency of events. In fact, frequency of events of 50 nucleotides single-strand DNA through aerolysin increases exponentially with temperature. At low voltage, the frequency is reaction-limited by an energy barrier of entry, and at high voltage, the frequency is diffusion-limited. They were able to extract an enthalpic barrier contribution of 15 k B T for the entrance of the oligonucleotide into the pore.
Time of transport was shown to decrease exponentially as a function of temperature ( Figure 3f). Furthermore, the authors were able to describe the free energy profile (Figure 3g) for the translocation of the oligonucleotides using a Fokker-Plank formalism. They found an unexpected large free-energy barrier for the threading stage (around 35 k B T) that can be attributed to the amplification of local pore-polymer attraction by the pore length. This result was not expected because the rate limiting step of polymer translocation was assumed to be a capture process.  (Figure 4f). Despite decades of multidisciplinary research on folding processes, during which an amino acid chain structures itself to become a functional protein, remains a challenge that has been able to be addressed with modern technics. In fact, many human diseases are related to partial unfolding or alternative folding 92,93 . These diseases result in reduced life expectancy and quality of life. In this context, the nanopore electrical detection could be a new instrumental and understanding the folding process. The ability to use a protein channel to probe protein denaturation in the presence of a chaotropic agent was first shown in 2007 with ⍺-hemolysin 94 .

Protein unfolding
An unfolding curve was extrapolated from current blockades frequencies (unfolded protein) and denaturant (guanidium) concentrations. The duration of the blockades depends on the conformation of the protein, unfolded or partially folded. In order to validate this novel method to study protein unfolding, prove of unfolding transition regardless the nanopore used was necessary. It was also crucial to compare single nanopore experiments to bulk experiments.
Aerolysin was chosen because the net charge and the geometry of the channel are different in comparison to ⍺-hemolysin nanopore. In the first example, we follow the protein stability, MalE219, a destabilized variant, as a function of temperature (figure 4A) 61 . Without protein, the current of the empty pore is stable at 25°C. After addition of proteins, we observe a few short spikes due to bumping events. An increase in temperature leads to an increase in number of spikes and their current amplitude (42°C). At higher temperature, we see the same behavior. In order to probe if these deep spikes are due to unfolded molecules, the normalized frequency of events is plotted against temperature. A sigmoidal relationship is found up to 50°C followed by a plateau and a surprising decrease in events. This sigmoidal behavior is attributed to an unfolding process because unfolded proteins can enter aerolysin. The duration of events decreases with temperature down to a plateau starting at about 60°C. This indicates all the events for unfolded protein at these temperatures cannot be observed due to their characteristic duration being too short to be resolved by the amplifier. In order to compare those results, a study was performed in bulk using circular dichroism with MalE219. A thermal sigmoidal unfolding curve with a long plateau is obtained up to 70°C (figure 4b). This curve confirms the resolution limit of the amplifier. Moreover, the transition temperatures are comparable between the bulk experiment (45.5 ± 0.5°C) and the single molecule approach (44 ± 1°C). In fact, the same unfolding transition is found with ⍺-hemolysin; the melting transition does not depend on the nanopore used. Another approach was to probe chemical denaturation of MalEwt, as a function of guanidium concentration at constant voltage ( Figure 4B). Extracted from the current traces (data not shown), the mean duration of short blockades is independent of the denaturant concentration used, <t duration > = 717 ± 39 µs. In other words, these events are due to unfolded proteins at the aerolysin channel entrance. The authors obtained a sigmoidal evolution of the frequency of events as a function of denaturing agent, up to a plateau value. The normalized frequency shows the same denaturation curve with an aerolysin or an ⍺-hemolysin nanopore (C half-aerolysin = 0.87 ± 0.01 M and C half-hemolysin = 0.85 ± 0.01 M.). For the long spike duration, a glassy behavior is found for aerolysin 91 and for ⍺-hemolysin nanopore 94 . These results demonstrated that the channel geometry and the net charge do not affect the unfolding transition or the first-order transition. On the other hand, it was observed that the event frequency and the dynamics of unfolded protein transport depend on the nanopore used 58,91,94 .  Recently, these authors have shown that this detection cannot be performed by an α-hemolysin channel because of a low confinement 96 . From the data obtained 70 , the inter-time T i distribution is plotted according to the number of di-saccharide units ( figure 6b). The inset of figure 6b shows the blockade frequency increases with the HA size, whereas a decrease was expected 97 . In figure   6c, the increase of the blockade current means increasing confinement in a manner consistent with previously reported with PEG chains and an α-hemolysin pore 98 . One may then suppose that detection is partially performed if the blockade duration is shorter than the rise time threshold (i.e. 2T r see Eq. 6). Figures 6d and 6e show an increase in blockade durations according to the number of units. In figure 6e, the corresponding characteristic times T c are bellow 2T r for hexaand octa-saccharides, showing that a part of the blockades is too short to be correctly detected.

Each distribution is fitted with a Gaussian function. Insert: average current blockade according to the number of monomeric units. The dotted line is a guide for the eyes. (d) Normalized logarithmic distribution of dwell time in presence of
This assumption is verified by the figure 6d where the duration distribution is truncated bellow the rise time threshold (dotted line in figure 6d). Aerolysin pore is therefore suitable for the detection of small oligosaccharides, but the transport duration must be increased account for all blockades. To perform this, Long and colleagues mutated binding sites inside the channel to increase nucleotide interactions 40 repeat units (PEG28, Mr = 1252) as an internal standard ( Figure 6B). They observed wellresolvable resistive pulses yielding a histogram of relative residual conductance (I/Io) with clearly discernible maxima corresponding to the different PEG species present. They found that, indeed, aerolysin mass spectrometer yields enhanced mass discrimination due to a combination of high signal to noise ratio as a consequence of the large driving force (electrophoretic owing to the coordination of cations to provide the intrinsically neutral PEG with a positive charge). In addition, they also found a steeper mass conductance relation and prolonged dwell times for smaller PEGs due to the pore geometry and its electrical properties.

Enzymatic degradation followed by electrical sensing
Enzymes are used in several biochemical reactions essential to the fields of biology and biomedical sciences. These reactions are characterized in bulk by spectrometry or by fluorescence. Nevertheless, the nanopore technique is an ideal tool to study these processes at a single molecule level 101 . Several publications focused on protein channel detection of enzymatically cleaved biomolecules (nucleotides 26 and peptides 102,103 ). Degradation of larger biologic nanoparticles such as amyloids can also be monitored by suitable track-etched channels 104 . α-hemolysin was mainly used to show nucleotide hydrolysis 26 , peptide cleavage 102,103 or the DNA ligase activity 105 . The high confinement of aerolysin is adapted to the detection of small molecules produced by the degradation of hyaluronic acid 70,74,96 or by the cleavage of neurotoxins 106 . In these publications, the authors performed the detection of an enzymatic degradation from the increase of blockade frequency. Just a few of them studied the enzyme kinetics at the single molecule scale by varying the substrate concentration according to a Michaelis−Menten model 74,106 . Long's group further explored the discrimination between oligonucleotides of different lengths 39 .

Oligonucleotide size and sequence discrimination
They showed that wt aerolysin exhibits excellent resolution for the discrimination of dA n (n = 2, 3, 4, 5 and 10) without any DNA labelling. The blockade currents histogram of a mixture of oligonucleotides show well defined pics for each length (Figure 7 A). The high current and temporal sensitivities are attributed to the geometry (constriction) and electrostatic interactions between the oligonucleotides and the pore. They also monitored the degradation of a dA 5 oligonucleotide by an ExoI restriction enzyme as a function of time, allowing them to separate the current levels of the cleavage intermediates. A detailed protocol for the discrimination of oligonucleotide lengths and real time monitoring of stepwise cleavage by ExoI is available 109 .
Structure of small oligonucleotides, from human telomeres, was investigated by Liao et al 110 .
Those oligonucleotides tend to fold and form G-quadruplexes which are impossible to detect through an aerolysin nanopore. The authors unfolded the oligonucleotides via a cation regulation mechanism (MgCl 2 ) and were able to detect different lengths of the telomeres. Dwell times were proportional to the length. Finally, Wang et al studied the effect of selectivity and sensitivity. They mutated the positively charged R220 at the entrance of the pore with negatively charged glutamic acid and by mutating the positively charged residue K238 with a glutamic acid 99 . They showed that transport of oligonucleotides was almost inhibited in the mutant R220E, mainly due to a higher entry barrier compared to the wt and demonstrating that the R220 is associated to entrance selectivity of the oligonucleotide into the pore. On the other hand, they showed an increase of the blockade times for the mutant K238E compared to the wt, due to strong interactions between the pore and the oligonucleotides, and demonstrating that K238 is associated to detection sensitivity of the oligonucleotides into the pore lumen.  Peptides are fundamental components of cells that key roles in regulating the activity of other molecules. Peptides consist in chains of amino acids that are linked between one another by amine bonds, similarly to proteins structure. However, peptides are defined as chains between 2 to ~100 amino acids. In addition, peptides tend to be less defined in structure than proteins, which can adopt complex conformations. Nevertheless, depending on their length and amino acid composition, peptides may fold as secondary structures, such as a-helix and b-sheets. Here we present how aerolysin has been used to study peptides. More than 10 years ago ⍺-hemolysin and aerolysin were the first biological nanopores used to probe ⍺-helix secondary structures. In their work 116 , they showed that different chemically modified neutral hetero-peptides, from 14 to 26 amino acids, are transported through the aerolysin nanopore. Transport of these ⍺-helical peptides were demonstrated to be of two types: a brief diffusion in the nanopore (type I), and the transport of the peptides inside the nanopore (type II). Both event types differ in terms of current blockade and blockade duration ( Figure 8A). A few years later, shorter peptides were analyzed, with the idea of demonstrating aerolysin nanopore potential for peptide/protein sequencing and biomarker detection. Long's group 117 showed the analysis of hetero-peptides that differ in terms of length (6 to 12 amino acids) and charge through the aerolysin particularly negatively charged peptides ( Figure 8B). Very recently, resolution of aerolysin has been extended to detection of short peptides of up to 3 amino acids long 32 . Homo-polymeric peptides of arginine residues (positively charged) from 3 to 10 amino acids were analyzed through the aerolysin nanopore. It appeared that these polycationic peptides are transported through the aerolysin nanopore. Their contribution to the current blockade is specific allowing for discrimination of peptides differing by just one single amino acid. Authors also compared peptides that differ in sequence per block of 5 amino acids and showed the discrimination of these peptides. These body of work highlights the exciting potential of aerolysin for peptide and protein sequencing, and, more broadly, proteomics.

DISCUSSION AND PERSPECTIVES
The discussion will focus only on the advantages and limits of aerolysin compared to other protein channels. It was previously observed that the main conditions for obtaining monomeric discrimination of polymer or peptide polydisperse solution using a protein channel are: (I) each monomer within a chain must contribute to the current blockade, this argument prevents an extended conformation inside the nanopore if the monomer number is larger than the persistence length (4 nucleotides for single stranded DNA and 2 amino-acids for a polypeptide chain); (II) a strong affinity between the molecule and the nanopore is necessary, the interaction's life time must be 15 to 20 smaller than the acquisition sampling time; (III) the protein channel must be size sensitive to the pore conductance change 32,37,38,118 . It has been shown that aerolysin has better size resolution for smaller polymer PEG chains 38 than an alpha-hemolysin nanopore 36 at high salt concentrations. The only difference is the applied potential which is higher with aerolysin (-120 mV) than alpha-hemolysin (+40 mV) and a signal-to-noise ratio increase of approximately 38 2.7.
The main factor is a considerable increase of the dwell times or interaction times between the small polymer chains and the aerolysin compared to the alpha-hemolysin. An increase in size resolution for larger PEG chains was demonstrated using alpha-hemolysin by increasing the temperature. It is due to the collapse of the polymers inside the pore 118 . For the longer chains, the resolution is progressively lost. Therefore, the first limit with these two sensors entails is observed when the radius of the macromolecule is larger than the pore radius. For higher molecular masses, the molecular weight distributions of a polydisperse polyelectrolyte (sodium polystyrene-sulfonate) solution can be evaluated by measuring chain translocation times through an α-hemolysin nanopore 119 . In this case, resolution lies in between a few kDa to a few dozens of kDa. Up to now, it has not been possible to obtain a monomeric resolution from a polydisperse solution. This phenomenon was also observed with single stranded DNA 39,40 and peptides 32 . The second limitation of aerolysin is the resolution of a single nucleotide 39 or 2 amino-acids 32 . This limit has not been reached with alpha-hemolysin for macromolecules and peptides 36,41,42,116,120 or with other protein channels 121,122 . In order to increase size resolution, one must increase the confinement by adding a molecular adaptor (modified cyclodextrin), decreasing the sensor diameter. This has been achieved for the detection of an individual nucleotide with an alphahemolysin pore 123 . A CuII-phenanthroline protein nanopore was designed to detect D-and Laromatic amino acids in order to force strong interactions between the monomer and the channel by chemical modification 124 . Another potential focus could involve finding aerolysin mutations to increase both current, amplitude and dwell time. Using selected mutants would potentially allow the discrimination of the 20 amino-acids, the main challenge being protein sequencing 6 .
Interest in detection of biomarkers from serum or cells using protein nanopores has increased and is reflected in recent publications. A true biomarker is a measurable characteristic indicator of normal biological and pathogenic processes utilized to assess the risk or presence of disease, pharmacological responses, or course of therapeutic intervention 125 . Only a few studies have been published on the detection of biomarkers model using protein nanopores. Most of those studies indirectly detect biomarkers by DNA assisted sensing [126][127][128] . To our knowledge, there are only a few articles on direct detection of biomarkers from serum for normal biological process 107,129 and human disease 34 . Up to now, aerolysin does not seem to be the perfect candidate for protein biomarker detection because of its geometry and narrow inner diameter.
Few publications on the nanopore field relate to protein unfolding. Up to now, this field remains a hot topic. Aerolysin and alpha-hemolysin are resistant to chaotropic agents 58,130 or temperature 60,131 . These resistant features made it possible to probe the stability of a wild type and a mutant protein by detecting partially unfolded or completely unfolded states. These resulted in the determination of single molecule level unfolding curves and the nature of phase transition 61,91,94 . Another approach was to study the co-translocational unfolding by electrical driving force 132,133 or with a molecular motor 30,134 using an alpha-hemolysin pore. A four-step unfolding mechanism during translocation was observed, for a thiorodoxin protein covalently attached to an oligonucleotide under a constant electrical driving force 132 . This differed from the mechanism observed in bulk. It was also shown that the unfolding process is different in term of molecular steps, kinetics and pathway if the pulling process is initiated by the protein C-terminus or N-terminus 133 . Another approach to study protein unfolding employs a molecular motor. An executed concept involved an ubiquitin-like protein (Smt3 domain) attached to a charged flexible linker with an ssrA tag at its C-terminus to bind an unfoldase ClpX variant. After the capture of the protein complex into the alpha-hemolysin and the binding to the motor, the unfoldase generated a mechanical force allowing unfolding of the protein. The current trace was different for the enzymatic unfolding process compared to the electrical pulling 30 . This allowed to identify protein domains (titin), variants and structural modifications 134 . A limit of the aerolysin is the possibility to detect and characterize peptides or small proteins in a label free approach, like Fragaceatoxin C, 122,129 Cytolisin A 135 or portal protein G20c channels 21 . The future challenges are to study disordered proteins involved in human diseases. It would also be interesting to perform unfolding curves of other proteins in order to determine their stability with ligands; and study their enzymatic unfolding with a molecular motor to probe secondary structures and topology domains.

VOCABULARY
Aerolysin: protein nanopore, member of the pore forming toxin (PFT's) family, expressed naturally by Pseudomonas aeruginosa. Nanopore: natural or artificial hole of nanometer size inside a membrane. Electrophoresis: The dynamics of an electrically charged molecule governed by an electric force. Electro-osmotic flow: movement of solvent across a charged nanopore induced by an applied electric potential. Energy barrier: an energy cost associated to ion/macromolecule entrance or transport through a nanopore. Size discrimination: the capability to separate macromolecules by their size/length at the monomer resolution using the blockade current amplitude.